论文部分内容阅读
目的应用环介导等温扩增(LAMP)技术建立马尔尼菲青霉菌(PM)快速检测方法,探讨该方法的特异性、敏感性等,评价其临床应用价值。方法针对马尔尼菲青霉菌的特异性基因MP1基因设计4条特异引物,反应前加入钙黄绿素荧光试剂,63℃恒温反应60min,结束后通过目视检测荧光及浊度仪检测产物浊度值判断结果;评价所建立LAMP方法的特异性和敏感性,建立标准曲线,并用LAMP法及PCR法检测样本,比较样本检出情况。结果该方法特异性良好,PM出现特异性LAMP扩增反应,而烟曲霉菌、腐皮镰刀菌、棘状外瓶酶菌、大肠埃希菌、嗜水气单胞菌、哈氏孤菌、创伤孤菌、水等均未出现扩增;检测限约为1.7×10-7 ng/μl,为PCR检测方法的10倍;以LAMP反应时间X轴,浓度的负次方数为Y轴,构建的标准曲线方程为y=0.4145x-8.9968,相关系数R2=0.9963,呈良好的线性关系;样本检测结果显示,LAMP法较PCR法获得更高的敏感性。结论 LAMP检测方法具有特异性高、敏感性高、反应时间短的特点,简便易行,适合于基层对PM的快速检测。
Objective To establish a rapid detection method for Penicillium marneffei (PM) using ring-mediated isothermal amplification (LAMP) technique and investigate the specificity and sensitivity of this method to evaluate its clinical value. Methods Four specific primers were designed for the MP1 gene of Penicillium marneffei. Calcein fluorescence reagent was added before the reaction and reacted at 63 ℃ for 60 min. At the end of the experiment, the turbidity value of the product was determined by visual detection of fluorescence and turbidimetry Results: The specificity and sensitivity of LAMP method was evaluated. The standard curve was established. The samples were detected by LAMP and PCR, and the detection of samples was compared. Results The specificity of this method was good, PM showed a specific LAMP amplification reaction, while Aspergillus fumigatus, Fusarium solani, Echinococcus, Escherichia coli, Aeromonas hydrophila, The detection limit was about 1.7 × 10-7 ng / μl, which was 10 times of the PCR detection method. Taking the LAMP reaction time on the X axis and the negative power of the concentration on the Y axis, The standard curve equation constructed was y = 0.4145x-8.9968, the correlation coefficient R2 = 0.9963, showing a good linear relationship; the sample test results showed that LAMP method was more sensitive than PCR method. Conclusion The LAMP detection method has the characteristics of high specificity, high sensitivity and short reaction time. It is simple and easy to operate and suitable for the rapid detection of PM in grass roots.